The integrity of the genome is continuously challenged by genotoxic agents and replication stress. Loss of genome stability can lead to pathological conditions such as cancer, premature aging, and neurodegeneration. Upon genomic alterations, cells coordinate a network of molecular pathways collectively known as the DNA damage response (DDR) that signals and promotes repair of DNA lesions, halting cell cycle progression until genome integrity is restored. Several lines of evidence indicate that genomic instability contributes to oncogenesis, cancer progression, and development of therapy resistance. Genetic analyses have identified high-, moderate-, and low-penetrance cancer susceptibility genes that are involved in DNA damage response and repair. Although many mechanistic and genetic studies have been performed over the years, a systematic analysis of the protein changes taking place on the chromosomes during the response to different types of genome perturbations is still lacking. High-resolution mass spectrometry-based proteomics is a robust method for the identification and quantification of proteins from complex mixtures. While affinity purification combined with mass spectrometry experiments have led to the discovery of intricate protein interaction networks, analyses of protein complexes assembled on chromatin have been much more challenging because purification methods are inefficient, biased or not compatible with mass spectrometry. However, this is rapidly evolving due to technological advances in proteomics. I propose to perform a comprehensive and unbiased quantitative and kinetic analysis of the protein landscapes assembled on fully functional nuclei and chromosomes during the response to different genotoxic agents, an approach that I call multiscale proteomics. Specifically, I will employ the cell-free extracts derived from the vertebrate Xenopus laevis eggs combined with state-of-the-art mass spectrometry analyses. This cell-free system allows experimental manipulations that cannot be achieved in cell systems and permits unprecedented characterization of the DNA damage response proteomes. I hypothesize that specific subsets of proteins recruited to chromatin under different damage conditions dictate not only the response to DNA damage, but also the usage of redundant repair pathways, which should shed some light on the occurrence of mutagenic forms of repair found in cancer genomes. Together with other members of the Gautier laboratory, I will validate and functionally characterize the findings in both Xenopus extracts and in human cells. Understanding how the protein networks that respond to and repair DNA damage work holds considerable potential to impact human health. From identifying useful synthetic lethal interactions that might enhance the efficacy of chemotherapy drugs to improving the safety and applicability of experimental gene therapies. Thus, we anticipate our studies will provide new insights on the regulation of the DNA damage responses, contributing to a better understanding of how the cells maintain the stability of their genomes upon genotoxic insults. !